Rho family small G protein activation has been implicated in the development of cancer and in metastatic progression. Rho protein activation is controlled by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs), so understanding the mechanisms regulating Rho GEF activity is critical to devising strategies to block Rho protein-mediated transformation. NET1 is a nuclear Rho GEF that is specific for the RhoA subfamily of small G proteins. Overexpression of NET1 results in its mislocalization in the cytoplasm, and stimulates constitutive RhoA activation and cell transformation. We have observed that NET1 isoforms are overexpressed in primary breast tumors, and that overexpression of NET1 proteins in breast cancer cell lines dramatically stimulates their proliferation. We have also observed that NET1 requires a C-terminal PDZ binding site to stimulate cell proliferation and transformation, and that this binding site mediates interaction with a protein complex consisting of the tumor suppressor Dlg1, the related protein Cask, and the DNA modifying enzyme Topo I. Since Dlg1 is a requisite target for oncoproteins from cancer causing viruses such as the human papilloma virus and the human T cell leukemia virus 1, the interaction between NET1 and Dlg1 is likely to be especially important to the mechanism by which NET1 controls cell proliferation. The hypothesis driving the proposed research is that overexpression of NET1 causes its mislocalization in the cytoplasm, and results in the constitutive activation of RhoA and an inhibition of the tumor suppressor function of Dlg1. We will address this hypothesis in the following specific aims. In Aim1 we will characterize the interaction of NET1 isoforms with Dlg1, Cask and Topo I in vitro and in breast epithelial cells. In Aim 2 we will elucidate how the phosphorylation of NET1 isoforms by regulatory kinases controls the interaction of NET1 with Dlg1. In Aim 3 we will examine the effects of NET1 overexpression, complex formation with Dlg1, Cask and Topo I, and NET1 phosphorylation on breast epithelial cell proliferation and transformation. Completion of these aims will delineate a unique and previously unrecognized role for NET1 and Dlg1 in controlling breast epithelial cell transformation, and identify potential new targets for intervention in this disease.